In a first phase of efforts, we discovered that the Tabby mouse, which has many of the features observed in human EDA, is specifically mutated in the corresponding mouse gene. We demonstrated that the Wnt pathway directly regulates EDA transcription. In published work, we further found that provision of DNA encoding a variant of ectodysplasin (Eda-A1, the longest isoform) in embryonic Tabby mice restores hair follicles and sweat glands. By generating Tet-regulated conditional transgenic mice, we have dissected spatiotemporal actions of Eda-A1 during hair follicle development. We also have characterized eye phenotypes of Tabby mice including blindness and inflammation susceptibility, and they are also reversed by supplementation with the same Eda-A1 isoform. This study provided the first animal model for ocular surface disease, and also further increased the interest in the possibility of manipulating the Eda pathway to combat dry eye. By large scale genome-wide expression profiling of samples from wild-type and Tabby mice ranging from embryos to adult and from hair follicles to sweat glands and primary keratinocytes, we identified numerous downstream target genes of Eda, including lymphotoxin-, Shh, Wnt10b and Dkk4 in hair follicles and Shh and FoxA1 in sweat glands. More recently, we have further focused on the function of Eda and Eda target genes identified by expression profiling in mutant mouse models. We demonstrated that target gene lymphotoxin-, an immune gene, is involved in hair shaft formation, but not hair follicle induction. We also found that Dkk4, a Wnt antagonist, discriminates an Eda-independent mechanism of secondary hair follicle formation. Notably, both pathways converge at the activation of downstream Shh. Based on these observation, we proposed that different subtypes of hair follicles are formed by variant molecular mechanisms. Conditional Shh transgenic mice and skin-specific Shh knockout mice in wild-type and Tabby backgrounds showed that Shh is required for elongation of Tabby hair shafts, but not for the induction of the primary hair follicles that are missing in Tabby mice. We have also studied and compared the control of Eda-independent skn appendage developmental pathways. A similar signaling pathway (TNF/NF-kB) is required for development of secondary lymphoid organs, but with very different downstream effectors. Skin appendage nails/claws is also independent of EDA, again regulated by a Wnt pathway early on, but working through Fzd6, the loss of Fzd6 distorted claw formation in mice, in line with the demonstrated damage of nail formation in patients lacking an active gene copy. We are now focusing on other skin exocrine glands, again aided by Tabby mice as a model. Genetic framework for sweat gland development. Analysis of cascade regulation of sweat gland development by major morphogenetic signaling pathways including Eda. Wnt/beta-catenin is required for Eda activation and the initiation of sweat gland formation. In Tabby mice, where Eda signaling is abolished, we found that sweat gland germs then started to form but aborted at pre-germ stage. Eda action is also required for sweat duct formation. Shh is located downstream of Eda, and required for final secretory portion formation. Further, a Wnt antagonist Dkk4 was located downstream of Wnt, and regulating sweat gland induction by a negative feedback effect on Wnt. Thus, we revealed a regulatory genetic framework Wnt-(Dkk4)-Eda-Shh for sweat gland development. This work was accepted for publication by Development recently. Additional layers of regulatory mechanism of sweat gland formation: Involvement of small non-coding RNAs in sweat gland development. miRNAs are known to act negatively for mRNA stabilities and their translation. To obtain better resolution of molecular mechanism, we are currently analyzing miRNA function in sweat gland development. We generated skin specific Dicer knockout mice, and found that sweat glands were not formed in the mutant mice. Expression profiling showed that an Egf family member, Epgn was significantly upregulated in the mutant mice. miRNA Q-PCR analysis further showed putative regulatory miRNAs of Epgn. We plan to generate skin specific Epgn transgenic mice to analyze Epgn and miRNA function in sweat gland development in detail. Secretory mechanism of sweat glands. The goal of this study is to analyze the mechanism of sweat secretion by identifying transcription factors and ion channels critical for initiation of sweat secretion. Previously, we carried out expression profiling with sweat gland samples from WT and Tabby that including developing and matured sweat glands.. Among genes expressed in sweat glands at different stages, FoxA1 was strikingly affected gene in Tabby during late developmental and adult stages. Skin-specific FoxA1 knockout mice showed striking anhidrosis, with abundant accumulation of glycoproteins in the lumens and ducts of otherwise complete sweat glands; and we further showed that FoxA1 functions in sweat glands by promoting transcription of an anion channel protein, Best2. Best2 knockout mice also showed severe hypohidrosis/anhidrosis, revealing a FoxA1-Best2 cascade as a fundamental genetic pathway in sweat glands, regulating sweat secretion. Because Best2 is a calcium activated bicarbonate channel, we inferred two alternative cascades for sweating: calcium K/Cl (FoxA1) additional monovalent ion transporter cascade and calcium (FoxA1) Best2 K/Cl ion transporter, with the latter likely playing the major role. We further found that Foxc1, another Fox family transcription factor, is also highly expressed in mouse sweat glands. To analyze Foxc1 function in sweat glands, we imported Foxc1-loxP mice recently from Dr. Kumes lab in Northwestern Univ., and generated skin specific Foxc1 knockout mice. In preliminary study, Foxc1 cKO mice showed severe hypohidrosis, however, sweat gland morphology was indistinguishable from WT controls. The phenotypes suggested that Foxc1 is not required for sweat gland development, but affects sweat secretion. We plan to identify affected genes in Foxc1 cKO sweat glands by expression profiling. We are also analyzing function of individual ion channels in sweat glands. K+ and Cl- channel opening is thought to be the first ionic event in sweat secretion. Analysis of the mechanism of meibomian gland development. Meibomian glands are specialized glands that lubricate the ocular surface. Tabby mice and EDA patients lack meibomian glands, and are thus susceptible for dry eye. We are characterizing the time and pattern of meibomian gland formation In Tabby and other models involving regulatory and signaling pathways. The effects and molecular targets of these pathways will be analyzed using the approaches we have established in our studies of other sweat glands. In addition, we are initiating a project Concerning another exocrine skin appendage, we previously showed that Eda-ablated Tabby mice develop ocular surface disease, and EDA patients show extreme dry eye. We plan to examine dry eye etiology with meibomian glands as an entry point. In a first phase, we characterized their development. They are missing in Tabby mice, and Shh knockout mice, Dkk4 transgenic mice, and beta-catenin knockout mice all completely lack meibomian glands. Currently we are characterizing meibomian gland phenotypes in these mice more systematically by time-course histological and immunohistochemical analyses, and assessing the possible trophic role of Eda in preventing aging-related deterioriation of Meibomian gland function. Toward skin appendages from embryonic stem cells. In further initiatives moving toward skin appendage regeneration, we have initiated an approach that could be more efficient starting from a master transcription factor that directs them toward keratinocytes.